Abstract
The Antarctic nematode Panagrolaimus sp. DAW1 is one of the only organisms known to survive extensive intracellular freezing throughout its tissues. Although the physiological mechanisms of this extreme adaptation are partly understood, the molecular mechanisms remain largely unknown. RNAi is a method that allows the examination of gene function in a direct, targeted manner, by knocking out specific mRNAs and revealing the effects on the phenotype. In this study we have explored the viability of RNAi in Panagrolaimus sp. DAW1. In the first trial, nematodes were fed E. coli expressing Panagrolaimus sp. DAW1 dsRNA of the embryonic lethal genes rps-2 and dhc, and the blister gene duox. Pd-rps-2(RNAi)-treated nematodes showed a significant decrease in larval hatching. However, qPCR showed no significant decrease in the amount of rps-2 mRNA in Pd-rps-2(RNAi)-treated animals. Several soaking protocols for dsRNA uptake were investigated using the fluorescent dye FITC. Desiccation-enhanced soaking showed the strongest uptake of FITC and resulted in a significant and consistent decrease of mRNA levels of two of the four tested genes (rps-2 and tps-2a), suggesting effective uptake of dsRNA-containing solution by the nematode. These findings suggest that RNAi by desiccation-enhanced soaking is viable in Panagrolaimus sp. DAW1 and provide the first functional genomic approach to investigate freezing tolerance in this non-model organism. RNAi, in conjunction with qPCR, can be used to screen for candidate genes involved in intracellular freezing tolerance in Panagrolaimus sp. DAW1.
Highlights
IntroductionDAW1 (formerly known as Panagrolaimus davidi CB1 and referred to here as PaDAW1 [1]) is the organism best documented to survive extensive intracellular freezing throughout its tissues [2]
We used the PaDAW1 and C. elegans homologues of rps-2 and dhc (Pd-rps-2 and Pd-dhc, and Ce-rps-2 and Ce-dhc-1 respectively) fed to both PaDAW1 and C. elegans to test whether a cross-species RNA interference (RNAi) effect could be detected similar to that reported in two other Panagrolaimus species and C. elegans [15]
Phenotypic analysis of embryonic lethal genes showed a significant reduction in larval hatching in Pd-rps-2(RNAi) treated PaDAW1 compared to the gfp(RNAi) treated controls (Fig 2A)
Summary
DAW1 (formerly known as Panagrolaimus davidi CB1 and referred to here as PaDAW1 [1]) is the organism best documented to survive extensive intracellular freezing throughout its tissues [2]. RNAi in the Antarctic Nematode Panagrolaimus sp. DAW1 observation of recrystallization inhibition and hexagonal ice crystals in extracts of PaDAW1 [7] imply the presence of ice active proteins [8,9]. A number of attempts have been made to isolate ice-active proteins expected to play a key role in the survival of intracellular freezing [7], including a recent molecular analysis providing a comprehensive transcriptome and draft genome [10]. Whereas the isolation and characterisation of any ice-active proteins has so far proven elusive, the molecular work has enabled the beginning of functional genomic work in the nematode. Environmental RNA interference (RNAi) is a method developed in C. elegans [11] that allows the functional role of genes to be explored by causing the degradation of targeted mRNA, but does not work in every nematode species [12]
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